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Chapter 16

DNA Damage, DNA Repair and Cancer

Carol Bernstein, Anil R. Prasad,Valentine Nfonsam and Harris Bernstein

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53919

1. Introduction

DNA damage appears to be a fundamental problem for life. In this chapter we review evi‐dence indicating that DNA damages are a major primary cause of cancer. DNA damagesgive rise to mutations and epimutations that, by a process of natural selection, can causeprogression to cancer. First, we describe the distinguishing characteristics of DNA damage,mutation and epimutation.

DNA damage is a change in the basic structure of DNA that is not itself replicated when theDNA is replicated. A DNA damage can be a chemical addition or disruption to a base ofDNA (creating an abnormal nucleotide or nucleotide fragment) or a break in one or bothchains of the DNA strands. When DNA carrying a damaged base is replicated, an incorrectbase can often be inserted opposite the site of the damaged base in the complementarystrand, and this can become a mutation in the next round of replication. Also DNA double-strand breaks may be repaired by an inaccurate repair process leading to mutations. In addi‐tion, a double strand break can cause rearrangements of the chromosome structure (possiblydisrupting a gene, or causing a gene to come under abnormal regulatory control), and, ifsuch a change can be passed to successive cell generations, it is also a form of mutation. Mu‐tations, however, can be avoided if accurate DNA repair systems recognize DNA damagesas abnormal structures, and repair the damages prior to replication. As illustrated in Figure1, when DNA damages occur, DNA repair is a crucial protective process blocking entry ofcells into carcinogenesis.

We note that DNA damages occur in both replicating, proliferative cells (e.g. those formingthe internal lining of the colon or blood forming “hematopoietic” cells), and in differentiat‐ed, non-dividing cells (e.g. neurons in the brain or myocytes in muscle). Cancers occur pri‐marily in proliferative tissues. If DNA damages in proliferating cells are not repaired due to

© 2013 Bernstein et al.; licensee InTech. This is an open access article distributed under the terms of theCreative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permitsunrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

inadequate expression of a DNA repair gene, this increases the risk of cancer. In contrast,when DNA damages occur in non-proliferating cells and are not repaired due to inadequateexpression of a DNA repair gene, the damages can accumulate and cause premature aging.As examples, deficiencies in DNA repair genes ERCC1 or XPF [1] or in WRN [2, 3] causeboth increased risk of cancer as well as premature aging. In Figure 1, DNA repair is indicat‐ed as a crucial process impeding both cancer and premature aging.

Figure 1. The roles of DNA damage and DNA repair in cancer and aging.

New Research Directions in DNA Repair414

A mutation is a change in the DNA sequence in which normal base pairs are substituted,added, deleted or rearranged. The DNA containing a mutation still consists of a sequence ofstandard base pairs, and the altered DNA sequence can be copied when the DNA is replicat‐ed. A mutation can prevent a gene from carrying out its function, or it can cause a gene to betranslated into a protein that functions abnormally. Mutations can activate oncogenes, inac‐tivate tumor suppressor genes or cause genomic instability in replicating cells, and an as‐semblage of such mutations, together in the same cell, can lead to cancer. Cancers usuallyarise from an assemblage of mutations conferring a selective advantage that leads to clonalexpansion (Figure 1). Colon cancers, for example, have an average of 15 “driver” mutations(mutations occurring repeatedly in different colon cancers) and about 75 “passenger” muta‐tions (mutations occurring infrequently in colon cancers) [4, 5]. Colon cancers also werefound to have an average of 9 duplications or deletions of chromosome segments [6] or,more recently, 17 focal amplifications, 28 recurrent deletions and up to 10 translocations [5].Since mutations have normal DNA structure, they cannot be recognized or removed byDNA repair processes in living cells. Removal of a mutation only occurs if it is sufficientlydeleterious to cause the death of the cell.

Another type of inheritable alteration, similar in some ways to a mutation, is an epige‐netic change. An epigenetic change refers to a functionally relevant modification of theDNA, or of the histone proteins controlling the relaxation or tightened winding of theDNA within their nucleosome structures. Some epigenetic changes involve specific altera‐tions of the DNA nucleotides. Examples of such changes include methylation of theDNA at particular sites (CpG islands) where the DNA starts to be transcribed into RNA.These changes may inhibit transcription. Other epigenetic changes involve modificationof histones associated with particular regions of the DNA. These may inhibit or promotethe ability of these regions to be transcribed into mRNA. Methylation of CpG islands ormodification of histones can directly alter transcription of gene-encoded mRNAs butthey can also occur in parts of the genome that code for microRNAs (miRNAs). MiRNAsare endogenous short non-protein coding RNAs (~22 nucleotides long) that post-tran‐scriptionally regulate mRNA expression in a sequence specific manner. miRNAs eithercause degradation of mRNAs or block their translation. Epigenetic modifications canplay a role similar to mutation in carcinogenesis, and about 280 cancer prone epigeneticalterations are listed by Schnekenburger and Diederich [7]. Epigenetic alterations are usu‐ally copied onto the daughter chromosomes when the parental chromosome replicates.

Although epigenetic changes can be passed down from one cell generation to the next,they are not regarded as true mutations. Most epigenetic changes appear to be part ofthe differentiation program of the cell and are necessary to allow different types of cellsto carry out different functions. In most cells of a human body, only about 5% of genesare active at any one time, often due to epigenetic modifications. However, abnormal un‐programmed epigenetic changes may also occur that alter the functioning of a cell andthese changes are referred to as “epimutations.” Programmed epigenetic changes can bereversed. During development, as daughter cells of a stem cell differentiate, some epige‐

DNA Damage, DNA Repair and Cancerhttp://dx.doi.org/10.5772/53919

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netic changes are programmed for reversal. However, a double strand break in DNA (atype of DNA damage) can initiate unprogrammed epigenetic gene silencing both bycausing methylation of a CpG island as well as by promoting silencing types of histonemodifications [8]. Another form of epigenetic silencing may occur during DNA repair.The enzyme Parp1 (poly(ADP)-ribose polymerase) and its product poly(ADP)-ribose(PAR) accumulate at sites of DNA damage as part of a repair process [9]. This, in turn,directs recruitment and activation of the chromatin remodeling protein ALC1 that maycause nucleosome remodeling [10]. Nucleosome remodeling has been found to cause, forinstance, epigenetic silencing of DNA repair gene MLH1 [11]. Chemicals previously iden‐tified as DNA damaging agents, including benzene, hydroquinone, styrene, carbon tetra‐chloride and trichloroethylene, were shown to cause considerable hypomethylation ofDNA, some through the activation of oxidative stress pathways [12]. Dietary agents alsohave been shown to affect DNA methylation or histone modification by numerous path‐ways [13]. Recent evidence indicates that epimutations occur in DNA repair genes thatreduce their function. Epimutations in DNA repair genes allow DNA damages to accu‐mulate, and are a cause of progression to cancer [14].

2. DNA damages are frequent, and DNA repair processes can beoverwhelmed

Tens of thousands of DNA damages occur per day per cell, on average, in humans, due toreactive molecules produced by metabolism or by hydrolytic reactions in the warm aqueouscellular media. Some types of such endogenous damages, and their rates of occurrence, areshown in Table 1.

A considerable number of other types of endogenous DNA damages have been identified,many of which are mutagenic. These include propano-, etheno- and malondialdehyde-de‐rived DNA adducts, base propenals, estrogen-DNA adducts, alkylated bases, deaminationof each of cytosine, adenine and guanine (to form uracil, hypoxanthine and xanthine, re‐spectively) and adducts formed with DNA by reactive carbonyl species [15].

While there are repair pathways that act on these DNA damages, the repair processes arenot 100% efficient, and further damages occur even as current DNA damages are being re‐paired. Thus there is a steady state level of many DNA damages, reflecting the efficiencies ofrepair and the frequencies of occurrence. For instance, Helbock et al. [16] estimated thesteady state level of oxidative adducts in rat liver as 24, 000 adducts per cell in young ratsand 66, 000 adducts per cell in old rats. Nakamura and Swenberg [17] determined the num‐ber of AP sites (apurinc and apyrimidinic sites) in normal tissues of the rat (i.e. in lung, kid‐ney, liver, testis, heart, colon and brain). The data indicated that the number of AP sitesranged from about 50, 000 per cell in liver, kidney and lung to about 200, 000 per cell in thebrain. These steady state numbers of AP sites in genomic DNA were considered to representthe balance between formation and repair of AP sites.


DNA damages Reported rate of occurrence Ref.

Oxidative

86,000 per cell per day in rats [18]

10,000 per cell per day in humans

100,000 per cell per day in rats[19]

11,500 per cell per day for humans

74,000 per cell per day for rats[16]

Specific oxidative damage products 8-

hydroxyguanine, 8-hydroxydeoxyguanosine, 5-

(hydroxymethyl) uracil

2,800 per cell per day in humans

34,800 per cell per day in mice[20]

Depurinations

10,000 per cell during 20-hour generation period [21]

13,920 per cell per day (580/cell/hr) [22]

2,000 to 10,000 per cell per day [23,24]

9,000 per cell per day [25]

Depyrimidinations

500 pyrimidines per cell during 20-hour generation

period[21]

696 per cell per day (29/cell/hr) [22]

Single-strand breaks 55,200 per cell per day (2,300/cell/hr) [22]

Double-strand breaks

~10 per cell cycle in humans [26]

~50 per cell cycle in humans [27]

O6-methylguanine 3,120 per cell per day (130/cell/hr) [22]

Cytosine deamination 192 per cell per day (8/cell/hr) [22]

Table 1. DNA damages due to natural endogenous causes in mammalian cells


417

DNA repair pathways are usually able to keep up with the endogenous damages in rep‐licating cells, in part by halting DNA replication at the site of damage until repair canoccur [28, 29]. In contrast, non-replicating cells have a build-up of DNA damages, caus‐ing aging [30, 31].

However, some exogenous DNA damaging agents, such as those in tobacco smoke, dis‐cussed below, may overload the repair pathways, either with higher levels of the same typeof DNA damages as those occurring endogenously or with novel types of damage that arerepaired more slowly. In addition, if DNA repair pathways are deficient, due to inheritedmutations or sporadic somatic epimutations in DNA repair genes in replicating somaticcells, unrepaired endogenous and exogenous damages will increase due to insufficient re‐pair. Increased DNA damages would likely give rise to increased errors of replication pastthe damages (by trans-lesion synthesis) or increased error prone repair (e.g. by non-homolo‐gous end-joining), causing mutations. Increased mutations that activate oncogenes, inacti‐vate tumor suppressor genes, cause genomic instability or give rise to other drivermutations in replicating cells would increase the risk of cancer.

3. Cancers are often caused by exogenous DNA damaging agents

Cancer incidence, in different areas of the world, varies considerably. Thus, the incidence ofcolon cancer among Black Native-Africans is less than 1 person out of 100, 000, while amongmale Black African-Americans it is 72.9 per 100, 000, and this difference is likely due to dif‐ferences in diet [32, 33]. Rates of colon cancer incidence among populations migrating fromlower-incidence to higher-incidence countries change rapidly, and within one generationcan reach the rate in the higher-incidence country. This is observed, for instance, in migrantsfrom Japan to Hawaii [34].

The most common cancers for men and women and their rates of incidence per 100, 000,averaged over the more developed areas and less developed areas of the world, are shownin Table 2 (from [35]). Overall, worldwide, cancer incidence in all organs combined is 300.1per 100, 000 per year in more developed areas and 160.3 per 100, 000 per year in less devel‐oped areas [35]. The differences in cancer incidence between more developed areas of theworld and less developed areas are likely due, in large part, to differences in exposure toexogenous carcinogenic factors. The lowest rates of cancers in a given organ (Table 2) maybe due, at least in part, to endogenous DNA damages (as described in the previous section)that cause errors of replication (trans-lesion synthesis) or error prone repair (e.g. non-homol‐ogous end-joining), leading to carcinogenic mutations. The higher rates (Table 2) are likelylargely attributable to exogenous factors, such as higher rates of tobacco use or diets higherin saturated fats that directly, or indirectly, increase the incidence of DNA damage.

It is interesting to note in Table 2 that, in cases where cancers occur in the same organs ofmen and women, men consistently have a higher rate of cancer than women. The basis forthis is currently unknown.


More developed areas Less developed areas

Incidence Mortality Incidence Mortality

Breast (women) 66.4 15.3 27.3 10.8

Prostate (men) 62.0 10.6 12.0 5.6

Lung (men) 47.4 39.4 27.8 24.6

Lung (women) 18.6 13.6 11.1 9.7

Colorectum (men) 37.6 15.1 12.1 6.9

Colorectum (women) 24.2 9.7 9.4 5.4

Esophagus (men) 6.5 5.3 11.8 10.1

Esophagus (women) 1.2 1.0 5.7 4.7

Stomach (men) 16.7 10.4 21.1 16.0

Stomach (women) 7.3 4.7 10.0 8.1

Liver (men) 8.1 7.2 18.9 17.4

Liver (women) 2.7 2.5 7.6 7.2

Bladder (men) 16.6 4.6 5.4 2.6

Bladder (women) 3.6 1.0 1.4 0.7

Cervix/Uterine (women) 12.9 2.4 5.9 1.7

Kidney (men) 11.8 4.1 2.5 1.3

Kidney (women) 5.8 1.7 1.4 0.8

Non-Hodgkin lymphoma (men) 10.3 3.6 4.2 3.0

Non-Hodgkin lymphoma

(women)7.0 2.2 2.8 1.9

Melanoma (men) 9.5 1.8 0.7 0.3

Melanoma (women) 8.6 1.1 0.6 0.3

Ovarian (women) 9.4 5.1 5.0 3.1

Table 2. Incidence and mortality rates for the most common cancers in age standardized rates per 100, 000 (excludingnon-melanoma skin cancer) (Adapted from Jemal et al. [35]).

4. Exogenous DNA damaging agents in carcinogenesis

Carcinogenic exogenous factors have been identified as a major cause of many common can‐cers, including cancers of the lung, colorectum, esophagus, stomach, liver, cervix/uterus andmelanoma. Often such exogenous factors have been shown to cause DNA damage, as de‐scribed below.


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5. Exogenous DNA damaging agents in lung cancer

In both developed and undeveloped countries, lung cancer is the most frequent causeof cancer mortality (Table 2, data for men and women combined). Lung cancer is large‐ly caused by tobacco smoke, since risk estimates for lung cancer indicate that, in theUnited States, tobacco smoke is responsible for 90% of lung cancers. Also implicated inlung cancer (and somewhat overlapping with smoking) are occupational exposure tocarcinogens (approximately 9 to 15%), radon (10%) and outdoor air pollution (perhaps 1to 2%) [36].

Acrolein 122.4

Formaldehyde 60.5

Acrylonitrile 29.3

1,3-butadiene 105.0

Acetaldehyde 1448.0

Ethylene oxide 7.0

Isoprene 952.0

Benzo[a]pyrene 0.014

Table 3. Weight, in μg per cigarette, of several likely carcinogenic DNA damaging agents in tobacco smoke (from [37]Cunningham et al., 2011])

Tobacco smoke is a complex mixture of over 5, 300 identified chemicals, of which 150 areknown to have specific toxicological properties (see partial summary by Cunningham [37]).A “Margin of Exposure” approach has recently been established to determine the most im‐portant exogenous carcinogenic factors in tobacco smoke [37]. This quantitative-type ofmeasurement is based on published dose response data for mutagenicity or carcinogenicityand the concentrations of these components in tobacco smoke (Table 3). Using the “Marginof Exposure” approach, Cunningham et al. [37] found the most important tumorigenic com‐pounds in tobacco smoke to be, in order of importance, acrolein, formaldehyde, acryloni‐trile, 1, 3-butadiene, acetaldehyde, ethylene oxide and isoprene.

Acrolein, the first agent in Table 3, is the structurally simplest α, β-unsaturated aldehyde(Figure 2). It can rapidly penetrate through the cell membrane and bind to the nucleophilicN2-amine of deoxyguanine (dG) followed by cyclization of N1, to give the exocyclic DNAadduct α-hydroxy-1, N2-propano-2’-deoxyguanine (α-HOPdG) (shown in Figure 2) and an‐other product designated γ-HOPdG. The adducts formed by acrolein are a major type ofDNA damage caused by tobacco smoke, and acrolein has been found to be mutagenic [38].

In tobacco smoke, acrolein has a concentration >8, 000 fold higher than benzo[a]pyrene (re‐viewed in [38]), with 122.4 μg of acrolein per cigarette. Benzo[a]pyrene has long beenthought to be an important carcinogen in tobacco smoke [39]. As reviewed by Alexandrov et


al. [39], benzo[a]pyrene damages DNA by forming DNA adducts at the N2 position of gua‐nine (similar to where acrolein forms adducts). However, by the “Margin of Exposure” ap‐proach, based on published dose response data and its concentration in cigarette smoke of0.014 μg per cigarette, benzo[a]pyrene is thought to be a much less important mutagen forlung tissue than acrolein and the other six highly likely carcinogens in tobacco smoke listedin Table 3 [37].

The other agents in Table 3 cause DNA damages in different ways. Formaldehyde, the sec‐ond agent in Table 3, primarily causes DNA damage by introducing DNA-protein cross-links. These cross-links, in turn, cause mutagenic deletions or other small-scalechromosomal rearrangements [40] and may also cause mutations through single-nucleotideinsertions [41]. Acrylonitrile, the third agent in Table 3, appears to cause DNA damage indi‐rectly by increasing oxidative stress, leading to increased levels of 8’-hydroxyl-2-deoxygua‐nosine (8-OHdG) in DNA [42]. Oxidative stress also causes lipid peroxidation that generatesmalondialdehyde (MDA), and MDA forms DNA adducts with guanine, adenine and cyto‐sine [43]. The fourth agent in Table 3, 1, 3-butadiene, causes genotoxicity both directly byforming a DNA adduct as well as indirectly by causing global loss of DNA methylation andhistone methylation leading to epigenetic alterations [44]. The fifth agent in Table 3, acetal‐dehyde, reacts with 2’-deoxyguanosine in DNA to form DNA adducts [45]. The sixth agentin Table 3, ethylene oxide, forms mutagenic hydroxyethyl DNA adducts with adenine andguanine [46]. The seventh agent in Table 3, isoprene, is normally produced endogenously byhumans, and is the main hydrocarbon of non-smoking human breath [47]. However, smok‐ing one cigarette causes an increase of breath isoprene levels by an average of 70% [48]. Iso‐prene, after being metabolized to mono-epoxides, causes DNA damage measured as singleand double strand breaks in DNA [49].

A large number of studies have been published in which the levels and characteristics ofDNA adducts in the lung and bronchus of smokers and non-smokers have been compared,as reviewed by Phillips [50]. In most of these studies, significantly elevated levels of DNAadducts were detected in the peripheral lung, bronchial epithelium or bronchioalveolar lav‐age cells of the smokers, especially for total bulky DNA adducts. As further discussed byPhillips [50], mean levels of DNA adducts in ex-smokers (usually with at least a 1 year inter‐val since smoking cessation) are found generally to be intermediate between the levels ofsmokers and life-long non-smokers. From these comparisons, the half-life of some DNA ad‐ducts in lung tissue are estimated to be ~1–2 years.

6. Exogenous DNA damaging agents in colorectal cancer

Up to 20% of current colorectal cancers in the United States may be due to tobacco smoke[51]. Presumably tobacco smoke causes colon cancer due to the DNA damaging agents de‐scribed above for lung cancer. These agents may be taken up in the blood and carried to or‐gans of the body.


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Figure 2. Reaction of acrolein with deoxyguanosine

The human colon is exposed to many compounds that are either of direct dietary origin or re‐sult from digestive and/or microbial processes. Four different classes of colonic mutageniccompounds were analysed by de Kok and van Maanen [52] and evaluated for fecal mutagenici‐ty. These included (1) pyrolysis compounds from food (heterocyclic aromatic amines and poly‐cyclic aromatic hydrocarbons), (2) N-nitroso-compounds (from high meat diets, from drinkingwater with high nitrates or produced during ulcerative colitis), (3) fecapentaenes (produced bythe colonic bacteria Bacteriodes in the presence of bile acids) and (4) bile acids (increased in thecolon in response to a high fat diet and metabolized to genotoxic form by bacteria in the colon).Many of these diet-related mutagenic compounds were analysed by Pearson et al. [53] in termsof their presence in fecal water, and their effect on the cytotoxic or genotoxic activity of fecalwater. Evidence in both of these studies was insufficient to evaluate the colorectal cancer risk asa result of specific exposures in quantitative terms.

However, substantial evidence implicates bile acids (the 4th possibility above) in colon caner.Bernstein et al. [54], summarized 12 studies indicating that the bile acids deoxycholic acid(DCA) and/or lithocholic acid (LCA) induce production of DNA damaging reactive oxygenspecies and/or reactive nitrogen species in colon cells of animal or human origin. They alsotabulated 14 studies showing that DCA and LCA induce DNA damage in colon cells. In ad‐dition to causing DNA damage, bile acids may also generate genomic instability by causing


mitotic perturbations and reduced expression of spindle checkpoint proteins, giving rise tomicro-nuclei, chromosome bridges and other structures that are precursors to aneuploidy[55]. Furthermore, at high physiological concentrations, bile acids cause frequent apoptosis,and those cells in the exposed populations with reduced apoptosis capability tend to surviveand selectively proliferate [54, 56]. Cells with reduced ability to undergo apoptosis in re‐sponse to DNA damage would tend to accumulate mutations when replication occurs pastthose damages, and such cells may give rise to colon cancers. In addition, 7 epidemiologicalstudies between 1971 and 1990 (reviewed by Bernstein et al. [54]), found that fecal bile acidconcentrations are increased in populations with a high incidence of colorectal cancer. Asimilar 2012 epidemiological study showed that concentrations of fecal LCA and DCA, re‐spectively, were 4-fold and 5-fold higher in a population at 65-fold higher risk of colon can‐cer compared to a population at lower risk of colon cancer [32]. This evidence points to bileacids DCA and LCA as centrally important DNA-damaging carcinogens in colon cancer.

Dietary total fat intake and dietary saturated fat intake is significantly related to incidence ofcolon cancer [57]. Increasing total fat or saturated fat in human diets results in increases inDCA and LCA in the feces [58, 59], indicating increased contact of the colonic epitheliumwith DCA and LCA. Bernstein et al. [60] added the bile acid DCA to the standard diet ofwild-type mice. This supplement raised the level of DCA in the feces of mice from the stand‐ard-diet fed mouse level of 0.3 mg DCA/g dry weight to 4.6 mg DCA/g dry weight, a levelsimilar to that for humans on a high fat diet of 6.4 mg DCA/g dry weight. After 8 or 10months on the DCA-supplemented diet, 56% of the mice developed invasive colon cancer.This directly indicates that DCA, a DNA damaging agent, at levels present in humans after ahigh fat diet, can cause colorectal cancer.

7. Exogenous DNA damaging agents implicated in other major cancers

It is beyond the scope of this chapter to detail the evidence implicating DNA damagingagents as etiologic agents in all of the significant cancers. Therefore, in Table 4 we indicatewith a single reference the major DNA damaging agent in five additional prevalent cancers,in order to illustrate the generality of exogenous DNA damaging agents as causes of cancer.In particular, we point out, as reviewed by Handa et al. [61], Helicobacter pylori infection in‐creases the production of reactive oxygen and reactive nitrogen species (RNS) in the humanstomach, which, in turn, significantly increases DNA damage in the gastric epithelial cells.Thus, H. pylori infection acts as a DNA damaging agent. In the case of human papillomavi‐rus (HPV) infection, Wei et al. [62] showed that cervical cells could resist RNS stress whennot infected with HPV. However, cervical cells infected by HPV and exposed to RNS hadhigher levels of DNA double strand breaks as well as a higher mutation rate. This appearedto occur due to the ability of HPV to greatly reduce protein expression of the DNA damagerepair/response gene P53 when infected cells were stressed by RNS. Since reduced P53 ex‐pression leads to greater RNS-induced DNA damage, HPV infection acts as a DNA damag‐ing agent in the presence of RNS stress.


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Cancer Exogenous DNA damaging agent Ref.

Esophagus Bile acids [63]

Stomach Helicobacter pylori infection [61]

Liver Aspergillus metabolite aflatoxin B(1) [64]

Cervix/UterusHuman papillomavirus plus increased nitric oxide from tobacco smoke or

other infection[62]

Melanoma UV light from solar radiation [65]

Table 4. Selected cancers and relevant implicated exogenous DNA damaging agents

8. Deficient DNA repair due to a germ line mutation allows DNAdamages to increase, leading to increased frequencies of mutation,epimutation and cancer

Expression of DNA repair genes may be reduced by inherited germ line mutations or genet‐ic polymorphisms, or by epigenetic alterations or mutations in somatic cells, and these re‐ductions may substantially increase the risk of cancer. Overall, about 30% of cancers areconsidered to be familial (largely due to inherited germ line mutations or genetic polymor‐phisms) and 70% are considered to be sporadic [66].

In 2 overlapping databases [67, 68] 167 and169 human genes (depending on the database)are listed that are directly employed in DNA repair or influence DNA repair processes. Thelists were originally devised by Wood et al. [69, 70]. The genes are distributed in groups ofDNA repair pathways and in related functions that affect DNA repair (Table 5). Bernstein etal. [71] illustrate many of the steps and order of action of the gene products involved for thefirst five DNA repair pathways listed in Table 5.

Individuals with an inherited impairment in DNA repair capability are often at considerablyincreased risk of cancer. If an individual has a germ line mutation in a DNA repair gene or aDNA damage response gene (that recognizes DNA damage and activates DNA repair), usu‐ally one abnormal copy of the gene is inherited from one of the parents and then the othercopy is inactivated at some later point in life in a somatic cell. The inactivation may be due,for example, to point mutation, deletion, gene conversion, epigenetic silencing or othermechanisms [72]. The protein encoded by the gene will either not be expressed or be ex‐pressed in a mutated form. Consequently the DNA repair or DNA damage response func‐tion will be deficient or impaired, and damages will accumulate. Such DNA damages cancause errors during DNA replication or inaccurate repair, leading to mutations that can giverise to cancer.

Increased oxidative DNA damages also cause increased gene silencing by CpG island hy‐permethylation, a form of epimutation. These oxidative DNA damages induce formationand relocalization of a silencing complex that may result in cancer-specific aberrant DNA


methylation and transcriptional silencing [73]. As pointed out above, the enzyme Parp1(poly(ADP)-ribose polymerase) and its product poly(ADP)-ribose (PAR) accumulate at sitesof DNA damage as part of a repair process [9], recruiting chromatin remodeling proteinALC1, causing nucleosome remodeling [10] that has been shown to direct epigenetic silenc‐ing of DNA repair gene MLH1 [11]. If silencing of genes necessary for DNA repair occurs,the repair of further DNA damages will be deficient and more damages will accumulate.Such additional DNA damages will cause increased errors during DNA synthesis, leadingto mutations that can give rise to cancer.

Number of genes listed in the two

databases

Homologous Recombinational Repair (HRR) 21,21

Non-homologous End Joining (NHEJ) 8,7

Nucleotide Excision Repair (NER) 30,29

Base Excision Repair (including PARP enzymes) (BER) 19,20

Mis-Match Repair (MMR) 11,10

Fanconi Anemia (FANC) [affects HRR (above) and translesion synthesis (TLS)] 10,16

Direct reversal of damage 3,3

DNA polymerases (act in various pathways) 17,15

Editing and processing nucleases (act in various pathways) 6,8

Ubiquitination and modification/Rad6 pathway including TLS 11,5

DNA damage response 12,14

Modulation of nucleotide pools 3,3

Chromatin structure 2,3

Defective in diseases and syndromes 4,5

DNA-topoisomerase crosslinks 2,1

Other genes 8,9

Table 5. DNA repair pathways and other processes affecting DNA repair [67, 68]


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9. Inherited mutations in genes employed in DNA repair that give rise tosyndromes characterized by increased risk of cancer.

Table 6 lists 36 genes for which an inherited mutation results in an increased risk of cancer.The proteins encoded by 35 of these genes are involved in DNA repair and in some casesalso in other aspects of the DNA damage response such as cell cycle arrest and apoptosis.The polymerase coded for by the 36th gene, XPV (POLH), is involved in bypass (rather thanrepair) of DNA damage, called translesion synthesis. The genes listed in Table 6, when mu‐tated in the germ line, give rise to a considerably increased lifetime risk of cancer, of up to100% (e.g. p53 mutations [74]). Thus defects in DNA repair cause progression to cancer.

In addition to mutations in genes that may substantially raise lifetime cancer risk, there appear tobe many weakly effective genetically inherited polymorphisms [single nucleotide polymo‐phisms (SNPs) and copy number variants (CNVs)]. By the HapMap Project, more than 3 millionSNPs have been found, and by Genome Wide Association studies (GWAs), about 30 SNPs werefound to increase risk of cancers. However the added risk of cancer by these SNPs is usuallysmall, i.e. less than a factor of 2 increase [75]. A large twin study [66], involving 44, 788 pairs oftwins, evaluated the risk of the same cancer before the age of 75 for monozygotic twins (identicalgenomes with the same polymorphisms) and dizygotic twins (having a 50% chance of the samepolymorphisms). If one twin had colorectal, breast or prostate cancer, the monozygotic twin hadan 11 to 18 percent chance of developing the same cancer while the dizygotic twin had only a 3 to9% risk. The differences in monozygotic and dizygotic rates of paired cancer were not significantfor the other 24 types of cancer evaluated in this study. Polymorphisms of the DNA repair geneERCC1 will be discussed below in relation to targeted chemotherapy.

10. Epimutations may repress DNA repair gene expression, allowingDNA damages to increase, leading to increased frequency of furtherepimutation, mutation and cancer

While germ line (familial) mutations in DNA repair genes cause a high risk of cancer, somat‐ic mutations in DNA repair genes are rarely found in sporadic (non-familial) cancers [4].Much more often, DNA repair genes are found to have epigenetic alterations in cancers.

One example of the epigenetic down-regulation of a DNA repair gene in cancers comes fromstudies of the MMR protein MLH1. Truninger et al. [76] assessed 1, 048 unselected consecutivecolon cancers. Of these, 103 were deficient in protein expression of MLH1, with 68 of these can‐cers being sporadic (the remaining MLH1 deficient cancers were due to germ line mutations). Ofthe 68 sporadic MLH1 protein-deficient colon cancers, 65 (96%) were found to be deficient due toepigenetic methylation of the CpG island of the MLH1 gene. Deficient protein expression ofMLH1 may also have been caused, in the remaining 3 sporadic MLH1 protein-deficient cancers(which did not have germ line mutations), by over expression of the microRNA miR-155. WhenmiR-155 was transfected into cells it caused reduced expression of MLH1 [77]. Overexpressionof miR-155 was found in colon cancers in which protein expression of MLH1 was deficient andthe MLH1 gene was neither mutated nor hypermethylated in its CpG island [77].


DNA repair gene(s)Encoded

protein

Repair pathway(s)

affectedRef.

Cancers with

increased riskRef.

breast cancer 1 & 2BRCA1,

BRCA2

HRR of double strand breaks

and daughter strand gaps[85] Breast, Ovarian [86]

ataxia telangiectasia mutated ATM

Different mutations in ATM

reduce HRR, single strand

annealing (SSA), NHEJ or

homology directed double

strand break rejoining

(HDR)

[87]Leukemia, Lymphoma,

Breast[87,88]

Nijmegen breakage syndrome NBS NHEJ [89] Lymphoid cancers [89]

meiotic recombination 11 MRE11 HRR and NHEJ [90] Breast [91]

Bloom’s Syndrome (helicase) BLM HRR [92]

Leukemia, Lymphoma,

Colon, Breast, Skin,

Auditory canal, Tongue,

Esophagus, Stomach,

Tonsil, Larynx, Lung,

Uterus

[93]

Werner Syndrome (helicase) WRN HRR, NHEJ, long patch BER [94]

Soft tissue sarcoma,

Colorectal, Skin,

Thyroid,

Pancreatic

[95]

Rothman Thomson syndrome

Rapadilino syndrome

Baller Gerold syndrome

RECQ4 Helicase likely active in HRR [96]

Basal cell carcinoma,

Squamous cell

carcinoma,

Intraepidemial

carcinoma

[97]

Fanconi’s anemia gene FANC

A,B,C,D1,D2,E,F,G,I,J,L,M,NFANCA etc. HRR and TLS [98]

Leukemia, Liver tumors,

Solid tumors many

areas

[99]

xeroderma pigmentosa

C, E [DNA damage binding

protein 2 (DDB2)]

XPC

XPE

Global genomic NER repairs

damage in both transcribed

and untranscribed DNA

[100,

101]

Skin cancer (melanoma

and non-melanoma)

[100,

101]

xeroderma pigmentosa

A, B, D, F, G

XPA XPB

XPD XPF

XPG

Transcription coupled NER

repairs the transcribed

strands of transcriptionally

active genes

[102]


and non-melanoma),

Central nervous system

cancers

[102]

xeroderma pigmentosa V (also

called polymerase H)

XPV

(POLH)Translesion Synthesis (TLS) [102]


and non-melanoma)[102]


427

DNA repair gene(s)Encoded

protein

Repair pathway(s)

affectedRef.

Cancers with

increased riskRef.

mutS (E. coli) homolog 2

mutS (E. coli) homolog 6

mutL (E. coli) homolog 1

postmeiotic segregation

increased 2 (S. cerevisiae)

MSH2

MSH6

MLH1

Pms2

MMR [76]Colorectal, endometrial.

ovarian[103]

mutY homolog (E. coli) MUTYHBER of A mispaired with

8OHdG, G, FapydG and C[104] Colon [105]

ataxia telaniectsia and RAD3

relatedATR

DNA damage response

likely affects HRR, not NHEJ[106] Oropharyngeal cancer [107]

Li Fraumeni syndrome P53

HRR, BER, NER and DNA

Damage Response for those

and for NHEJ and MMR

[108]

Sarcoma, Breast, Lung,

Skin, Pancreas,

Leukemia, Brain

[74]

Table 6. Inherited mutations in DNA repair genes that increase the risk of cancer

Another example of the epigenetic down-regulation of a DNA repair gene in cancer comes fromstudies of the direct reversal of methylated guanine bases by methyl guanine methyl transferase(MGMT). In the most common form of brain cancer, glioblastoma, the DNA repair gene MGMTis epigenetically methylated in 29% [78] to 66% [79] of tumors, thereby reducing protein expres‐sion of MGMT. However, for 28% of glioblastomas, the MGMT protein is deficient but theMGMT promoter is not methylated [79]. Zhang et al. [78] found, in the glioblastomas withoutmethylated MGMT promoters, that the level of microRNA miR-181d is inversely correlatedwith protein expression of MGMT and that the direct target of miR-181d is the MGMT mRNA3’UTR (the three prime untranslated region of MGMT mRNA), though they indicated that othermiRNAs may also be involved in the reduction of protein expression of MGMT.

Almost all DNA repair deficiencies found, so far, in sporadic cancers, and in precanceroustissues surrounding cancers (field defects) are due to epigenetic changes. Examples of suchepigenetic alterations in DNA repair genes in different types of cancer are shown in Table 7.A recent review [80] lists 41 reports (mostly not overlapping with those listed in Table 7)indicating methylation of 20 DNA repair genes in various cancers. In Table 7 data are alsoshown on DNA repair gene deficiencies for the field defects associated with colorectal, gas‐tric, laryngeal and non-small cell lung cancer.

As summarized above, epimutations can result from oxidative DNA damages. Such damagescause formation and relocalization of a silencing complex that in turn causes increased gene si‐lencing by CpG island hypermethylation [73]. Epigenetic nucleosome remodeling during DNArepair can also silence gene expression [11]. When CpG island methylation or nucleosome re‐modeling or other types of epigenetic alterations (e.g. micro RNAs or histone modifications) in‐hibit DNA repair genes, more damages will accumulate. Accumulated DNA damages causeincreased errors during DNA synthesis and repair. Thus epigenetic deficiencies in DNA repairgenes can have a cascading effect (a mutator phenotype), leading to genomic instability and ac‐cumulation of mutations and epimutations that can give rise to cancer.


Cancer

Epigenetic changes

in cancer

(mechanism)

% sporadic cancers

with epimutations

Epigenetic

changes in field

defect

(mechanism)

% field defects

with epi-

mutations

Ref.

BreastBRCA1 (CGI*)

13% unselected

[108]67% medullary

55% mucinous

WRN (CGI) 17% unselected [2]

Ovarian BRCA1 (CGI)

31% of those with

loss of

heterozygosity

[108]

Colorectal

WRN (CGI) 38% [2]

MGMT (CGI) 46% MGMT (CGI) 23% [109]

MGMT (CGI) 90%[110]

MLH1 (CGI) 65%

MLH1 (CGI) 10% [76]

MLH1 (CGI) 2%

[111]MSH2 (CGI) 13% MSH2 5%

MGMT (CGI) 47% MGMT 11%

ERCC1 100% ERCC1 40%

[112]PMS2 88% PMS2 50%

XPF 55% XPF 40%

GastricMGMT (CGI) 88% MGMT (CGI) 29% [113]

WRN (CGI) 25% [2]

Esophageal

squamous cell

carcininoma

MLH1 (CGI) 49%

[114,115]MLH2 (CGI) 35%

MGMT (CGI) 41%

Larynx MGMT (CGI) 54% MGMT (CGI) 38% [116]


429

Cancer

Epigenetic changes

in cancer

(mechanism)

% sporadic cancers

with epimutations

Epigenetic

changes in field

defect

(mechanism)

% field defects

with epi-

mutations

Ref.

Non-small cell LungWRN (CGI) 38% [2]

MGMT (CGI) 70% MGMT (CGI) 40% [117]

Prostate WRN (CGI) 20% [2]

Thyroid WRN (CGI) 13% [2]

Non-Hodgkin

lymphomaWRN (CGI) 24% [2]

Leukemias WRN (CGI) 5-10% [2]

Chondrosarcomas WRN (CGI) 33% [2]

Osteosarcomas WRN (CGI) 11% [2]

Brain glioblastomaMGMT (CGI) 51% [118]

MGMT (miRNA) 28% [78]

Liver hepatocellular

carcinoma

P53 (non-CGI

promoter site specific

methylation)

100% [119]

Papillary thyroid

(tested 23 DNA repair

genes for CGI)

MLH1 (CGI) 21%

[120]PCNA (CGI) 13%

OGG1 (CGI) 2%

*CGI=CpG island methlyation

Table 7. Examples of epigenetic alterations (epimutations) of DNA repair genes in cancers and in field defects, withmechanisms indicated where known.

Deficiencies in DNA repair genes cause increased mutation rates. Mutations rates increasein MMR defective cells [81, 82] and in HRR defective cells [83]. Chromosomal rearrange‐ments and aneuploidy also increase in HRR defective cells [84]. Thus, deficiency in DNA re‐pair causes genomic instability and genomic instability is the likely main underlying causeof the genetic alterations leading to tumorigenesis. Deficient DNA repair permits the acquis‐ition of a sufficient number of alterations in tumor suppressor genes and oncogenes to fuelcarcinogenesis. Deficiencies in DNA repair appear to be central to the genomic and epige‐nomic instability characteristic of cancer.


Figure 3 illustrates the chain of consequences of exposure of cells to endogenous andexogenous DNA damaging agents that lead to cancer. The role of germ line defects inDNA repair genes in familial cancer are also indicated. The large role of DNA damageand consequent epigenetic DNA repair defects leading to sporadic cancer are empha‐sized. The roles of germ line mutation and directly induced somatic mutation in spora‐dic cancer are indicated as well.

Figure 3. The roles of DNA damage, epigenetic deficiencies in DNA repair and mutation in progression to cancer.


431

11. Epigenetic alterations caused by micro RNAs

MicroRNAs (miRNAs) are endogenous non-coding RNAs, 19-25 nucleotides in length, thatcan have substantial effects on DNA repair. miRNAs can either directly or indirectly reduceexpression of DNA repair or DNA damage response genes. As discussed above, over-ex‐pression of miR-155 causes reduced expression of DNA repair protein MLH1, and miR-155is overexpressed in colon cancers [77] (curved arrow in Figure 3). Similarly, miR-181d isoverexpressed in glioblastomas, causing reduced expression of DNA repair protein MGMT[78]. Although miRNAs can epigenetically regulate DNA repair gene expression, the expres‐sion levels of many miRNAs may themselves be subject to epigenetic regulation. One mech‐anism of epigenetic regulation of miRNA expression is hypomethylation of the promoterregion of the DNA sequence that codes for the miRNA. Schnekenburger and Diederich [7]list miR-155 as one of a long list of mi-RNAs whose expression is increased by hypomythy‐lation in colorectal cancers. In particular, hypomethylated miR-155 (the hypomethylationmaking it more active) targets genes MLH1, MSH2 and MSH6, causing each of them to havereduced expression [7].

Wan et al. [121] referred to 6 further DNA repair genes that are directly targeted by miR‐NAs. ATM, RAD52, RAD23B, MSH2, BRCA1 and P53, are each specifically targeted by oneor two of the 8 miRNAs miR-21, miR-24, miR-125b, miR-182, miR-210, miR-373, miR-421and miR-504, with all but miR-210, miR-421 and miR-504 among those identified by Schne‐kenburger and Diederich [7] as overexpressed through epigenetic hypomethylation. Overex‐pression of any one of these miRNAs leads to reduced expression of its target DNA repairgene. Wan et al. [121] further listed 16 DNA damage response genes targeted by specificmiRNAs. Wan et al. [121] indicated miR-15a, miR-16, miR-17, miR-20a, miR-21, miR-24,miR-29, miR-34a, miR-106a, miR-93, miR-124a, miR-125b, miR-192, miR-195, miR-215,miR-182, miR-373 as among those targeting DNA damage response genes. Of these, all butmiR-124a were identified by Schnekenburger and Diederich [7], (and Malumbres [122] fur‐ther identified miR-34a and miR-124a) as being among miRNAs whose expression is subjectto epigenetic alteration in tumors. Other miRNAs whose expression is subject to epimuta‐tion in colorectal cancers (and their target DNA repair or DNA damage response genes) in‐clude miR-17 (E2F1), miR-34b/c (P53), miR-106a (E2F1), miR-200a and miR-200b (MLH1,MSH2) and miR-675 (Rb) [7].

12. Epigenetic alterations caused by chromosome remodeling and histonemodification

Specific miRNAs can also indirectly (and strongly) reduce protein expression of DNA repairgenes through their role in repression of proteins designated High Mobility Group A1(HMGA1) and HMGA2 (the names come from the proteins’ high electrophoretic mobility onacrylamide gels). HMGA1 and HMGA2 cause chromatin remodeling at specific sites inDNA and reduce expression at those sites. In particular, these proteins appear to control


DNA repair genes BRCA1 and ERCC1. BRCA1 And ERCC1 proteins have key roles in DNArepair, particularly of double-strand breaks and interstrand crosslinks. HMGA1 and HMGA2genes are usually active in embryogenesis, but normally have very low expression levels inadult tissues. Their expression levels in adult tissues are kept low by the actions of specificmiRNAs. If expression of these miRNAs is reduced, then the repressive HMGA1 andHMGA2 proteins become highly expressed and, in particular, can reduce expression ofBRCA1 or ERCC1 respectively.

As reviewed by Resar [123], all HMG proteins share an acidic carboxyl terminus and asso‐ciate with chromatin. As an example, HMGA1A, in particular, has three AT-hook domainsthat allow it to bind to AT-rich regions and recruit an “enhanceosome” that may displacehistones and cause chromosome remodeling and reduce gene expression. Baldassarre et al.[124] showed that HMGA1B protein binds to the promoter region of BRCA1 and inhibitsBRCA1 promoter activity (indicated in Figure 3 as chromatin remodeling causing reducedBRCA1). In 12 surgically removed human breast carcinomas, there was an inverse correla‐tion between HGMA1 protein and BRCA1 mRNA levels. HGMA1 was almost undetectablein normal breast tissue, highly expressed in the tumor samples, and BRCA1 protein wasstrongly diminished in tumor samples. Baldassarre et al. [124] suggested that while only11% of breast tumors had hypermethylation of the BRCA1 gene, 82% of aggressive breastcancer specimens have low BRCA1 protein, and most of these could be due to chromatin re‐modeling by high levels of HMGA1 protein.

Similarly, HMGA2 binds to an ERCC1 promoter site and represses ERCC1 promoter activity[125]. The miRNAs miR-23a, miR-26a and miR-30a inhibit HMG2A protein expression [126]though it has not been reported whether these miRNAs are under epigenetic control. In Fig‐ure 3, one of two dotted lines is used to indicate possible repression of ERCC1 by epigeneti‐cally induced chromatin remodeling.

Resar [123] and Baldassarre et al. [124] summarized reports indicating that HGMA1 is wide‐ly overexpressed in aggressive malignancies including cancers of the thyroid, head andneck, colon, lung, breast, pancreas, hematopoetic system, cervix, uterine corpus, prostateand central nervous system. Palmieri et al. [127] showed that HGMA1 and HMGA2 are tar‐geted (and thus strongly reduced in expression) by miR-15, miR-16, miR-26a, miR-196a2 andLet-7a. The promoter regions associated with miR-16, miR-196a2 and Let-7a miRNAs are ep‐imutated by hypomethylation [7, 122] while Sampath et al. [128] showed, in addition, thatthe coding regions for miR-15 and miR-16 were epigenetically silenced due to histone deace‐tylase activity. Palmieri et al. [127] further showed that these 5 miRNAs are drastically re‐duced in a panel of 41 pituitary adenomas, accompanied by increases in HMGA1 andHMGA2 specific mRNAs. In a more recent study on pituitary adenomas by D’Angelo et al.[129], reduced expression of 18 miRNAs was found, with 5 of them targeting HMGA1 orHMGA2. In this recent study, among the 18 miRNAs with reduced expression, the reducedexpression of miR-26b, miR-34b, miR-432 and miR-592 was known to be due to epigeneticalteration [7, 122]. Thus, epigenetic miRNA silencing, causing strong expression of HMGA1and HMGA2, occurs in many types of cancer and this may be related to reductions found inexpression of DNA repair genes BRCA1, BRCA2 and ERCC1.


433

Suzuki et al. [130], using genome wide profiling, found 174 primary transcription units formiRNAs, called “pri-miRNAs” (large precursor RNAs which may encode multiple miR‐NAs), of which they identified 37 as potential targets for epigenetic silencing. Of these 37pri-miRNAs, 22 were encoded by DNA sequences with CpG islands (all of which were hy‐permethylated in colorectal cancer cells) while the other pri-miRNAs were subject to regula‐tion by epigenetic “activating marks” without evidence of deregulated methylation.

Activating marks are alterations on histones that cause transcriptional activation of thegenes associated with those altered histones (reviewed by Tchou-Wong et al. [131]). In par‐ticular, the nucleosome, the fundamental subunit of chromatin, is composed of 146 bp ofDNA wrapped around an octamer of four core histone proteins (H3, H4, H2A, and H2B).Posttranslational modifications (i.e., acetylation, methylation, phosphorylation, and ubiqui‐tination) of the N- and C-terminal tails of the four core histones play an important role inregulating chromatin biology. These specific histone modifications, and their combinations,are translated, through protein interactions, into distinct effects on nuclear processes, suchas activation or inhibition of transcription. In eukaryotes, methylation of lysine 4 in histoneH3 (H3K4), which interacts with the promoter region of genes, is linked to transcriptionalactivation. There is a strong positive correlation between trimethylation of H3K4, transcrip‐tion rates, active polymerase II occupancy and histone acetylation. Thus trimethylation ofH3K4 is an activating mark.

In addition to pri-miRNAs being regulated by activating marks, some miRNAs appear to bedirectly regulated by these histone modifications. As summarized by Sampath et al. [128],histone deacetylases catalyze the removal of acetyl groups on specific lysines around genepromoters to trigger demethylation of otherwise methylated lysine 4 on histones(H3K4me2/3) and this causes loss of these activating marks, promoting chromatin compac‐tion, and leading to epigenetic silencing. Sampath et al. [128] showed that such histone de‐acetylase activity mediates the epigenetic silencing of miRNAs miR-15a, miR-16, andmiR-29b. As indicated above, miR-15, miR-16 specifically target HGMA1 and HMGA2. IfmiR-15 and miR-16 lose their activating marks, they have reduced expression, causingHGMA1 and HGMA2 to be transcriptionally activated, thus reducing expression of DNA re‐pair genes BRCA1 and ERCC1.

In Figure 3, histone modification and chromatin remodeling are indicated as epigenticallyaltering the expression of many genes in progression to cancer, and specifically causing re‐duced BRCA1 and possibly (as indicated by one dotted line) reduced expression of ERCC1.In addition, a second dotted line is used to indicate possible repression of ERCC1 by an miR‐NA. Klase et al. [132] showed that a particular virally coded miRNA down regulates ERCC1protein expression at the p-body level (a p-body is a cytoplasmic granule “processing body”that interacts with miRNAs to repress translation or trigger degradation of target mRNAs).A survey of human miRNA homology regions to ERCC1 mRNA indicates at least 21 humancoded miRNAs that could act to decrease ERCC1 mRNA translation (shown in MicrocosmTargets [133]). ERCC1 protein expression, assessed by immunohistochemical staining, is de‐ficient due to an epigenetic mechanism in colon cancers [110], and this could be due to ac‐tion of one or more miRNAs, acting directly on ERCC1 mRNA.


13. Driver mutations and pathways to cancer progression

Recent research indicates a mechanism by which an early driver mutation may cause subse‐quent epigenetic alterations or mutations in pathways leading to cancer. Wang et al. [134]point out that isocitrate dehydrogenase genes IDH1 and IDH2 are the most frequently mu‐tated metabolic genes in human cancer. A gene frequently mutated in cancer is consideredto be a driver mutation [4] so that mutations in IDH1 and IDH2 would be driver mutations.Wang et al. [134] further point out that IDH1 and IDH2 mutant cells produce an excess met‐abolic intermediate, 2-hydroxyglutarate, which binds to catalytic sites in key enzymes thatare important in altering histone and DNA promoter methylation. Thus, mutations in IDH1and IDH2 generate a DNA CpG island methylator phenotype that causes promoter hyper‐methylation and concomitant silencing of tumor suppressor genes such as the DNA repairgenes MLH1, MGMT and BRCA1. As shown in Figure 3, a driver mutation in IDH1 cancause a feedback loop leading to increased DNA repair deficiency, further mutations andepimutations, and consequent accelerated tumor progression.

A study, involving 51 patients with brain gliomas who had two or more biopsies over time,showed that mutation in the IDH1 gene occurred prior to the occurrence of a p53 mutationor a 1p/19q loss of heterozygosity, indicating that IDH1 mutation is an early driver mutation[135]. Work by Turcan et al. [136] showed that IDH1 mutation alone is sufficient to establishthe brain glioma CpG island methylator phenotype. Carillo et al. [137] showed that when anIDH1 mutation was present in glioblastoma tumors, 64% of these were hypermethylated inthe promoter regions of MGMT.

Other initial driver mutations can cause progression to glioblastoma as well. As pointed outabove, increased levels of miR-181d also cause reduced expression of MGMT protein in glio‐blastoma. Nelson et al. [138] indicate that a single type of miRNA may target hundreds ofdifferent mRNAs, causing alterations in multiple pathways. Patients with a glioblastomathat does not harbor an IDH1 mutation have an overall fairly short survival time, while pa‐tients with both mutated IDH1 and methylated MGMT have a subtype of glioblastoma witha much longer survival time (implying a different pathway of cancer progression) [137].

An IDH1 mutation that gives rise to a CpG island methylator phenotype that causes pro‐moter hypermethylation and concomitant silencing of MGMT also causes promoter silenc‐ing of other genes as well. In addition to silencing of genes, the CpG island methylatorphenotype can cause methylation of the promoter regions of long interspersed nuclear ele‐ment-1 (LINE-1) DNA sequences. Ohka et al. [139] point out that LINE-1 is a class of retro‐posons that are the most successful integrated mobile elements in the human genome, andaccount for about 18% of human DNA. Ohka et al. [139] found that LINE-1 methylation isdirectly proportional to MGMT promoter methylation in gliomas and suggested that LINE-1methylation could be used as a proxy to indicate the CpG island methylator phenotype sta‐tus in glioblastomas. This phenotype, likely associated with methylation of the MGMT pro‐moter, in turn, indicates whether treatment with the DNA alkylating agent temozolomidewill be beneficial in treatment of a patient with a glioblastoma, since MGMT removes thealkyl groups added to guanine by temozolomide.


435

14. Field defects

Field defects have been described in many types of gastrointestinal cancers [140]. A field de‐fect arises when an epimutation or mutation occurs in a stem cell that causes that stem cellto give rise to a number of daughter stem cells that can out-compete neighboring stem cells.These initial mutated cells form a patch of somewhat more rapidly growing cells (an initialfield defect). That patch then enlarges at the expense of neighboring cells, followed by, atsome point, an additional mutation or epimutation arising in one of the field defect stemcells so that this new stem cell with two advantageous mutations can generate daughterstem cells that can out-compete the surrounding field defect of cells that have just one ad‐vantageous mutation. As illustrated in Figure 4, this process of expanding sub-patches with‐in earlier patches will occur multiple times until a particular constellation of mutationsresults in a cancer (represented by the small dark patch in Figure 4. It should also be notedthat a cancer, once formed, continues to evolve and continues to produce sub clones. A renalcancer, sampled in 9 areas, had 40 ubiquitous mutations, 59 mutations shared by some, butnot all regions, and 29 “private” mutations only present in one region [141].

.

Figure 4. Schematic of a field defect in progression to cancer


.

Figure 5. Colon resection including a colon cancer. Dashed arrows indicate grossly unremarkable colonic mucosa. Ul‐cerated hemorrhagic mass represents a moderately differentiated invasive adenocarcinoma. Solid arrow indicates theheaped up edge of the malignant ulcer

Figure 5 shows an opened resected segment of a human colon that has a colon cancer. Asillustrated by Bernstein et al. [142], there are about 100 colonic microscopic epithelial cryptsper sq mm in the colonic epithelium. The resection shown in Figure 5 has an area of about6.5 cm by 23 cm, or 150 sq cm, or 15, 000 sq mm. Thus this area has about 1.5 million crypts.There are 10-20 stem cells at the base of each colonic crypt [143, 144]. Therefore there arelikely about 15 million stem cells in the grossly unremarkable colonic mucosal epitheliumshown in Figure 5. Evidence reported by Facista et al. [112], and listed in Table 7, indicatesthat in many such resections, most of the stem cells in such an area up to 10 cm distant (ineach direction) from a colon cancer (such as in the grossly unremarkable area shown in Fig‐ure 5), and the majority of their differentiated daughter cells, are epigenetically deficient forprotein expression of the DNA repair genes ERCC1, PMS2 and/or XPF, although the epithe‐lium is histologically normal.

The stem cells most distant from the cancer, deficient for ERCC1, PMS2 and/or XPF, can beconsidered to constitute an outer ring, and be deficient as well in the inner rings, of a fielddefect schematically illustrated in Figure 4. The outer ring in Figure 4 includes, within itscircumscribed area, on the order of 15 million stem cells, presumably arising from an initialprogenitor stem cell deficient in DNA repair (due to epigenetic silencing). As a result of thisrepair deficit, the initial stem cell was genetically unstable, giving rise to an increased fre‐quency of mutations in its decendents. One daughter stem cell among its decendents had amutation that, by chance, provided a replicative advantage. This descendent then under‐went clonal expansion because of its replicative advantage. Among the further decendentsof the clone, new mutations arose frequently, since these descendents had a mutator pheno‐type [145], due to the repair deficiency passed down epigenetically from the original repair-defective stem cell. Among these new mutations, some would provide further replicativeadvantages, giving rise to a succession of more aggressively growing sub clones (innerrings), and eventually a cancer.


437

15. Exogenous carcinogenic agents cause reduced expression of DNArepair genes

Many known carcinogenic agents cause reduced expression of DNA repair genes or directlyinhibit the actions of DNA repair proteins. Table 8 lists examples of carcinogens that havesuch effects. Due to space limitations, many other such carcinogens are not listed. Thesefindings further link DNA damage to cancer.

Carcinogens Inhibit DNA Repair Gene Mechanism Shown Ref.

Arsenic compounds

PARP [146,147]

XRCC1

[146,148]Ligase 3

Ligase 4

DNA POLB, XRCC4[146]

DNA PKCS, TOPO2B

OGG1, ERCC1, XPF [149]

XPB, XPC, XPE [150]

P53Inhibition of P53 serine 15

phosphorylation[151]

Cadmium compounds

MSH2, ERCC1, XRCC1Promoter methylation [152]

OGG1

MSH2, MSH6 proteins Cd2+ binds to proteins [153]

OGG1 protein Oxidation of Ogg1 [154]

DNA-PK, XPD [155]

XPC [156]

Bile acids

deoxycholateMUTYH, OGG1 mRNA reduced [157]

BRCA1 [158]

lithocholate DNA POLB [159]

Lipid peroxidation

compounds

4-hydroxy-2-nonenal (4-HNE ) Nuc. Excision Repair NER protein adducts [160]

Malondialdehyde Nuc. Excision Repair NER protein adducts [161]


Carcinogens Inhibit DNA Repair Gene Mechanism Shown Ref.

Oxidative stress

MisMatch RepairOxidative damage to MMR

proteins[162]

ERCC1 protein Oxidative attack [163]

OGG1 protein Degraded by calpain [164]

Gamma irrad. OGG1, XRCC1 mRNA reduced [165]

Benzo(a)pyrene BRCA1 miR-638 increased [166]

Methylcholanthrene/

diethylnitrosamineBRCA1, ERCC1, XRCC1, MLH1 [167]

Styrene XRCC1, OGG1, XPC mRNA reduced [168]

Aristolochic acid P53, PARP1, OGG1, ERCC1, MGMT mRNA reduced [169]

Antimony XPE mRNA reduced [170]

Nickel MGMT Promoter methylation [171]

Table 8. Examples of carcinogenic agents that cause reduced expression of DNA repair genes

16. Polyphenols can epigenetically increase expression of DNA repairgenes

Some polyphenols affect expression of many genes, including DNA repair genes, throughepigenetic alterations, as reviewed by Link et al. [172]. Examples of DNA repair genes ex‐pression increased by epigenetic alteration are listed in Table 9.

Phytochemical Plant source MechanismTargeted DNA

Repair GenesRef.

Epigalocatechin-3-gallate Green teaReversal of CpG island

methylationMGMT, MLH1 [173]

Dihydrocoumarin Yellow sweet clover p53 acetylation P53 [174]

Genistein SoyReversal of CpG island

methylationMGMT [173]

Genistein Soy Histone acetylation P53 [175]

Table 9. Examples of phytochemicals that increase expression of DNA repair genes by an epigenetic mechanism


439

17. Possible protection against cancer by phytochemicals that increaseDNA repair by unknown mechanisms

A recent review article by Collins et al. [176] summarizes some examples of micronutrients thataffect DNA repair gene expression, though by unknown mechanisms. Table 10 lists such phy‐tochemicals, without defined mechanisms, that increase DNA repair gene expression, alongwith commonly known foods that are high in those phytochemicals [177, 178, 179].

Phytochemical (test system) Examples of foods high in nutrient

Increased DNA

Repair Gene

Expression

Ref.

Ellagic acid (mice) Raspberries, pomeganateXPA, ERCC5, DNA

Ligase 3[180]

Silymarin (cells in vitro) Artichoke, milk thistle MGMT[181]

Curcumin (cells in vitro) Turmeric MGMT

Chlorogenic acid (cells in vitro)Blueberries, coffee, sunflower seeds,

artichokePARP

[182]

Caffeic acid (cells in vitro) coffee, cranberry, carrot PMS2

m-coumaric acid (cells in vitro) olives (and metabolite of caffeic acid) PARP, PMS2

3-(m-hydroxyphenyl) propionic acid (cells

in vitro)

(major metabolite of caffeic acid and

degradation product of proanthocyanidins

in chocholate)

PARP, PMS2

Table 10. Examples of phytochemicals that increase expression of DNA repair genes by unknown mechanisms

Bernstein et al. [182] evaluated antioxidants based on their ability to increase DNA repairproteins PARP-1 and Pms2 in vitro. They tested 19 anti-oxidant compounds and of these 19compounds only chlorogenic acid and its metabolic products: chlorogenic acid, caffeic acid,m-coumaric acid and 3-(m-hydroxyphenyl) propionic acid, increased expression of the twotested DNA repair genes in HCT-116 cells (Table 10).

Chlorogenic acid (CGA) (high in blueberries, coffee, sunflower seeds, artichoke) [177, 183,184] was then tested as a preventive agent in the recently devised diet-related mouse modelof colon cancer [60]. As described above in the section Exogenous DNA damaging agents incolorectal cancer, deoxycholic acid (DCA), a DNA damaging agent, at levels present after ahigh fat diet, can cause colorectal cancer. When DCA is added to the diet of wild-type miceto raise the level of DCA in the mouse feces to the level in feces of humans on a high fat diet,by 10 months of feeding 94% of the mice develop tumors in their colons with 56% develop‐ing colonic adenocarcinomas [60]. This mouse model develops tumors solely in the colon,phenotypically similar to development of colon cancer in humans. When CGA, equivalentto 3 cups of coffee a day for humans, was added to the DCA supplemented diet it was dra‐


matically protective against development of colon cancer, reducing incidence of colon can‐cer significantly from 56% to 18% [60].

18. Targeting of chemotherapeutic agents to cancers deficient in DNArepair

As discussed above, DNA repair deficiency often arises early in progression to cancer andcan give rise to genomic instability, a general feature of cancers. If cancer cells are deficientin DNA repair they are likely to be more vulnerable than normal cells to inactivation byDNA damaging agents. This vulnerability of cancer cells can be exploited to the benefit ofthe patient. Some of the most clinically effective chemotherapeutic agents currently used incancer treatment are DNA damaging agents, and their therapeutic effectiveness appears tooften depend on deficient DNA repair in cancer cells.

In the next four sections we discuss repair deficiencies in cancer cells that can be effectivelytargeted by DNA damaging chemotherapeutic agents. In addition, deficiency in a DNA re‐pair pathway that arises during tumor development may make cancer cells more reliant ona remaining reduced set of DNA repair pathways for survival. Recent studies indicate thatdrugs that inhibit one of these alternative pathways in such cancers cells can be useful incancer therapy. Targeting cancer cells having a repair deficiency with specific DNA damag‐ing agents, or with agents that inhibit alternative repair pathways, offers a new promisingapproach for treating a variety of cancers.

19. Targeting cancers deficient in BRCA1

The BRCA1 (breast cancer 1 early onset) protein is employed in an important DNA repairpathway, homologous recombinational repair (HRR). This pathway removes a variety oftypes of DNA damages, and is the only pathway that can accurately remove double-stranddamages such as double-strand breaks and inter-strand cross-links. BRCA1 also has otherfunctions related to preservation of genome integrity (reviewed by Yun and Hiom [185]). In‐dividuals with a germ-line inherited defect in the BRCA1 gene are at increased risk of breast,ovarian and other cancers. In addition to inherited germ-line defects in BRCA1, deficienciesin expression of this gene may arise in somatic cells either by mutation or by epimutationduring progression to sporadic (non-germline) cancer.

Patients with a variety of types of cancer are treated effectively with chemotherapeuticagents that cause double-strand breaks (e.g. the topoisomerase inhibitor etoposide), or causeinter-strand cross-links (e.g. the platinum compound cisplatin). These damages can causecancer cells to undergo apoptosis (a form of cell death). However, patients treated withthese agents often prove to be intrinsically resistant, or develop resistance during treatment.Quinn et al. [186] demonstrated that BRCA1 expression is necessary for such resistance. Thisfinding suggests that BRCA1-mediated DNA repair can protect cancer cells from therapeutic


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DNA damaging drugs. Thus, although high expression of BRCA1 may be initially beneficialto the individual by reducing the risk of developing cancer, it also may be detrimental oncecancer has developed by counteracting the therapeutic effect of DNA-damaging agents tar‐geted to the cancer cells.

Patients with non-small cell lung cancer (NSLC) are often treated with DNA cross-link‐ing platinum therapeutic compounds such as cisplatin, carboplatin or oxaliplatin. NSCLCis the leading cause of cancer deaths worldwide, and almost 70% of patients withNSCLC have locally advanced or metastatic disease at diagnosis. Improved survival afterplatinum-containing chemotherapy in metastatic NSCLC correlates with low BRCA1 ex‐pression in the primary tumor [187, 188]. This finding indicates that low BRCA1-mediat‐ed DNA repair is detrimental to the cancer upon treatment, and thus beneficial to thepatient. BRCA1 likely protects cancer cells by participating in a pathway that removesthe potentially lethal DNA cross-links introduced by the platinum drugs. Since lowBRCA1 expression in the tumor appears to be beneficial to the patient, Taron et al. [187]and Papadek et al. [188] concluded that BRCA1 expression is potentially an importanttool for use in cancer management and should be assessed for predicting chemosensitivi‐ty and tailoring chemotherapy in lung cancer.

Over 90% of ovarian cancers appear to arise sporadically in somatic cells and are associatedwith BRCA1 dysfunction. Weberpals et al. [189] showed for patients having sporadic ovari‐an cancer treated with platinum drugs, the median survival was longer for patients withlower expression of BRCA1 vs. higher BRCA1 expression (46 vs. 33 months).

20. Targeting cancers deficient in ERCC1

ERCC1 (Excision Repair Cross-Complementaion group 1) is a key protein needed to re‐move platinum adducts and repair inter- and intra-strand cross-links [190]. ERCC1 di‐merizes with XPF (xeroderma pigmentosum complementation group F) protein to form acomplex that can excise damaged DNA. Over-expression of ERCC1 is associated withcellular resistance to platinum compounds, whereas ERCC1 down-regulation sensitizescells to cisplatin [191, 192].

Cisplatin has made a major impact in the chemotherapeutic treatment of testicular cancer.Over 90% of patients with newly diagnosed testicular germ cell cancer, and 70 to 80% of pa‐tients with metastatic testicular cancer, can be cured using cisplatin based combination che‐motherapy [193]. Hypersensitivity of testicular cancer to cisplatin appears to be due to lowlevels of the three NER proteins ERCC1, XPF and XPA [194].

Simon et al. [195] evaluated ERCC1 mRNA expression in lung tumors as a predictor of sur‐vival of NSCLC patients. They found that patients with relatively low ERCC1 mRNA ex‐pression had poor overall survival. This finding suggests that low ERCC1-mediated DNArepair allows DNA damages to persist and give rise to carcinogenic mutations. However,they also noted that those NSCLC tumors with relatively low ERCC1 expression responded


better to platinum based therapy. Lord et al. [196] found that low ERCC1 mRNA expressionin the primary tumor correlates with prolonged survival after cisplatin plus gemitabine che‐motherapy in NSCLC. Median overall survival with low ERCC1 expression tumors was 61.6weeks compared to 20.4 weeks for patients with high expression tumors.

Zhou et al. [197] reported that a particular genetic polymorphism that alters ERCC1 mRNAlevel predicts overall survival in advanced NSCLC patients treated with platinum basedchemotherapy. Olaussen et al. [198] found that patients with completely resected NSCLC tu‐mors that were ERCC1-negative benefited from adjuvant cisplatin-based chemotherapy,whereas patients with ERCC1-positive tumors did not benefit. They suggested that determi‐nation of ERCC1 expression in NSCLC cells before chemotherapy can make a contributionas an independent predictor of the effect of adjuvant chemotherapy. Papadaki et al. [188]found that ERCC1 mRNA level in the primary tumor of patients with metastatic NSCLCcould predict the effectiveness of cisplatin based chemotherapy. Low ERCC1 mRNA levelwas significantly associated with higher response rate, longer median progression-free sur‐vival and median overall survival. Leng et al. [199] found that patients with ERCC1 negativeexpression had a longer progression free survival and overall survival than ERCC1 positivepatients after receiving platinum based adjuvant therapy. Thus ERCC1 mRNA level, likeBRCA1 mRNA level (discussed above), in the primary tumor at the time of diagnosis couldbe used to predict platinum sensitivity of NSCLC.

ERCC1 expression also appears to have predictive significance for ovarian cancer. Dab‐holkar et al. [200] found in ovarian tumor tissues that ERCC1 mRNA expression levelswere higher in patients who were resistant to platinum based therapy than in those pa‐tients who responded to such therapy. Kang et al. [201] observed that a particular poly‐morphism of the ERCC1 gene sequence was associated with clinical outcome of platinumbased chemotherapy in patients with ovarian cancer. Weberpals et al. [189] also showedfor ovarian cancer patients that higher ERCC1 mRNA level, alone, or especially in com‐bination with higher BRCA1 mRNA level in the tumor, predicted shorter overall patientsurvival after platinum therapy.

ERCC1 protein expression is often reduced within colon cancers and in a field defect sur‐rounding these cancers [112]. For metastatic colorectal cancer patients receiving combinationoxaliplatin and fluorouracil chemotherapy, lower ERCC1 mRNA expression in the tumorpredicts longer survival [202]. Viguier et al. [203] found that a particular ERCC1 geneticpolymorphism predicts a better tumor response to oxaliplatin/5-fluorouracil combinationchemotherapy in patients with metastatic colorectal cancer.

Low ERCC1 mRNA levels also predict better response and survival for gastric cancer pa‐tients [204] and bladder cancer patients [205] receiving cisplatin-based chemotherapy.

Thus numerous studies involving cancer of the testis, lung, ovary, colon, stomach and blad‐der indicated that platinum based chemotherapy can enhance patient outcome when target‐ed specifically to tumors with low ERCC1 expression. Such tumors have diminished abilityto repair the DNA damages, particularly the cross-links, induced in the tumors by the plati‐num compound.


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21. Targeting cancers deficient in MGMT

Alkylating agents, including chloroethylnitrosoureas, procarbazine and temozolomide, arecommonly used to treat malignant brain tumors. These agents cause DNA damage by add‐ing alkyl groups to DNA. Such damages may then be repaired or, if unrepaired, trigger celldeath. As an example, temozolomide methylates DNA at several sites generating mainly N7-methylguanine and N3-methyladenine adducts, which constitute nearly 90% of the totalmethylation events. However these adducts are efficiently removed and accurately replacedby the base excision repair pathway, and thus have low cytotoxic potential. About 5 to 10%of the methylation events caused by temozolomide produce O6-methylguanine which is cy‐totoxic, and this adduct accounts for the beneficial therapeutic effect of temozolomide andother alkylating agents on malignant brain tumors.

O6-methylguanine methyltransferase (MGMT) is a DNA repair enzyme that rapidly re‐verses alkylation (including methylation) at the O6 position of guanine, thus neutraliz‐ing the cytotoxic effects of chemotherapeutic alkylating agents such as temozolomide.High MGMT activity in tumor tissue is associated with resistance to alkylating agents.MGMT activity is controlled by a promoter sequence, and methylation of the CpG is‐land in the promoter silences the gene in cancer cells, so that these cells no longer pro‐duce MGMT. In addition, as described above, an increased level of miR-181d can alsodecrease MGMT expression and help the ability of temozolomide to give a beneficialtherapeutic effect [78].

Esteller et al. [206] showed that methylation of the MGMT promoter increases the re‐sponsiveness of the gliomas (brain tumors) to chemotherapeutic alkylating agents, lead‐ing to regression of the tumors and prolonged overall and disease free survival. Paz etal. [207] showed that hypermethylation of CpG islands within the promoter sequence ofthe MGMT gene predicts a better clinical response to temozolomide in primary gliomas.They considered that their results might open up possibilities for more customizedtreatments of human brain tumors. Hegi et al. [208] demonstrated a significantly im‐proved clinical outcome in patients with malignant glioma who had a methylatedMGMT promoter and were treated with temozolomide. The 18-month survival rate was62% among patients with a methylated MGMT promoter compared with only 8% in theabsence of promoter methylation. Hegi et al. [209] reviewed further evidence thatMGMT promoter methylation is associated with improved progression-free and overallsurvival in malignant glioma patients treated with alkylating agents. They also dis‐cussed strategies to overcome MGMT-mediated chemoresistance that are currently un‐der investigation. Upon reviewing the relevant evidence, Weller et al. [210] concludedthat MGMT promoter methylation is the key mechanism of MGMT gene silencing, andcould be used as a biomarker for predicting a favorable outcome in patients with ma‐lignant glioma who are exposed to alkylating chemotherapy. They considered that thisbiomarker is on the verge of entering clinical decision-making.


22. Targeting cancers with a repair deficiency using a PARP inhibitor;synthetic lethality

If a tumor is deficient in an essential protein component of a DNA repair pathway, the can‐cer cells would likely be more reliant on remaining DNA repair pathways for survival.Drugs that inhibit one of these alternative pathways, in principle, might prove to be usefulin cancer therapy by selectively killing the cancer cells. An example of such an approach isthe use of poly(ADP-ribose) polymerase [PARP] inhibitors against tumors that are deficientin BRCA1 or BRCA2 [211]. This approach has provided proof-of-concept for an anticancerstrategy termed “synthetic lethality.” By this strategy the inhibition of a particular repairpathway in cancer cells that are already deficient in another repair pathway preferentiallyinduces greater toxicity in repair deficient cancer cells than in normal non-cancer cells. Cur‐rent research guided by this strategy is directed at finding new agents that inactivate proteincomponents of major repair pathways, and thus could be targeted against cancers that arealready deficient in another repair pathway [212].

A germ-line mutation in one BRCA1 or BRCA2 allele substantially increases the risk ofdeveloping several cancers, including breast, ovarian, and prostate cancer. Diploid cellsheterozygous for either a BRCA1 or a BRCA2 mutant allele may lose expression of theremaining wild-type allele, resulting in deficient homologous recombinational repair.This loss causes an increase in unrepaired DSBs that can lead to mutations (throughcompensatory inaccurate repair) and chromosomal aberrations that drive carcinogenesis.Inactivation of the wild-type allele in the cell lineage leading to the tumor is thought tobe an obligate step in this carcinogenesis pathway, a step that does not occur in thenormal non-cancer tissues of the patient.

The deficiency in homologous recombinational repair is thus specific to the tumor, and canbe exploited by employing PARP inhibitors. Ordinarily, single-strand breaks (SSBs), as dis‐tinct from DSBs, are repaired by the base excision repair pathway, in which the enzymePARP1 plays a key role. The inhibition of PARP1 leads to the accumulation of DNA SSBs.Unrepaired SSBs can give rise to DSBs at replication forks during DNA replication. ThusPARP inhibition in tumor cells with deficient homologous recombinational repair (becauseof the absence of BRCA1 or BRCA2) generates unrepaired SSBs that are likely to cause anoverwhelming accumulation of DSBs leading to tumor cell death. In contrast, the normal tis‐sues of a patient consists of cells that are heterozygous for a BRCA1 or BRCA2 mutant alleleand therefore retain homologous recombinational repair function, and have a sensitivity toPARP inhibitors similar to that of wild-type cells. Thus PARP inhibition induces selective tu‐mor cell killing while sparing normal cells.

Fong et al. [213] conducted a preliminary clinical evaluation of the oral PARP inhibitorolaparib. They observed that 63% of patients carrying BRCA1 or BRCA2 mutations whohad ovarian, breast or prostate cancer had a clinical benefit from treatment with olapar‐ib with few adverse side effects. This is an example of the concept of “synthetic lethali‐ty” which occurs when there is a potent lethal synergy between two otherwise non-


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lethal events. The two events in this case are (1) a specific PARP inhibitor blocks repairof SSBs causing an increase in SSBs leading to an increase in DSBs; and (2) a tumor re‐stricted genetic loss of function or homologous recombinational repair that is ordinarilyneeded to accurately repair these DSBs.

A subsequent trial of olaparib in BRCA mutation-associated breast cancer demonstrated ob‐jective positive response rates of 41%, again with limited toxicity [214]. About 10% of wom‐en with ovarian cancer carry a BRCA1 or BRCA2 mutant allele. Audeh et al. [215] showedthat the oral PARP inhibitor olaparib has antitumor activity in women carriers of BRCA1 orBRCA2 alleles who have ovarian cancer. The objective positive response rate was 33%.

23. Overview of the role of DNA damage and repair in carcinogenesis

In this section we present a brief overview of the relationship of DNA damage and re‐pair to carcinogenesis, and the implications of this relationship for strategies of preven‐tion and therapy, emphasizing the evidence reviewed above. Carcinogenesis is generallyviewed as a Darwinian process that occurs in a somatic cell lineage by mutation or epi‐mutation and natural selection. Natural selection operates on the basis of the adaptivebenefit to individual cells in the lineage of more rapid cell division or higher resistanceto cell death (apoptosis) than occurs in neighboring cells. Most of the random mutationsand epimutations that arise during progression to cancer are likely to be disadvanta‐geous or neutral from the prospective of the emerging cancerous cells, and only thosethat promote more rapid overall growth are advantageous. The cell lineage that ultimate‐ly becomes a cancer probably passes through a series of evolutionary pre-cancerousstages involving sequential rounds of mutation/epimutation and selection [216]. The ini‐tial stage is probably a lineage of cells with a small selective advantage that forms anearly field within a tissue. Within this defective field successive mutation and selectionevents occur which finally give rise to an invasive and then metastatic cell lineage. Dur‐ing this process the cell lineage acquires the hallmarks of cancer (summarized by Hana‐han and Weinberg [217]). These include: sustaining proliferative signaling, evadinggrowth suppressors, resisting cell death, enabling replicative immortality, inducing angio‐genesis, reprogramming energy metabolism, and evading immune destruction.

Mutations arise from unrepaired DNA damages, either by translesion synthesis duringDNA replication or by inaccurate repair of DNA damages, as in the inaccurate process ofnon-homologous end joining of double-strand breaks. Mutations may also arise by sponta‐neous replication errors without the intervention of DNA damage, but this source of muta‐tion is likely less frequent than mutations caused by DNA damage. The primary cause(s) ofepimutations (such as CpG island methylations) are not well understood, but evidence sug‐gests that epimutations arise during the repair processes that remove DNA damages. Thesources of DNA damage underlying carcinogenesis can be extrinsic or intrinsic. Epidemio‐logic evidence suggests that a large proportion of the DNA damages contributing to cancerarise from extrinsic stressful conditions, including such factors as smoking, high fat diet, cer‐


tain infections and UV light exposure. The possible contribution from intrinsic causes, suchas free radical production during normal metabolism, have not been assessed. A pervasivecharacteristic of human tumors is genomic instability [217]. A likely major source of this in‐stability is loss of DNA repair capability. Germ line mutations in DNA repair genes general‐ly lead to syndromes characterized by a greatly increased risk of cancer. The majority ofcancers arise sporadically, i.e. are not primarily due to germ line mutations. A frequent char‐acteristic of sporadic cancers is loss of expression of one or more DNA repair proteinsthrough epigenetic silencing. The several different DNA repair pathways that occur in mam‐malian cells each specialize in removing different types of damage, but they are also partial‐ly overlapping. Thus reduction of a particular repair pathway may have differentcarcinogenic consequences from loss of another repair pathway [218]. However, the deleteri‐ous effect of loss of one pathway may be partially ameliorated by another functioning path‐way.

This general view of the role of DNA damage and repair in carcinogenesis has implicationsfor the prevention and treatment of cancer. Cancer incidence could be substantially reducedby a general avoidance of the known sources of DNA damage such as smoking. In additionto avoiding DNA damage, it should also be beneficial to increase DNA repair, or at least toavoid extrinsic factors that decrease repair. The factors affecting repair capability are lesswell studied than those causing DNA damage, but several are known, and a significant ben‐efit may be derived from considering such factors as well.

The finding that DNA repair deficiency is a common feature of cancers, and is perhaps theunderlying cause of the genetic instability of cancers, has implications for therapy. If a can‐cer is composed of cells deficient in DNA repair, it is, in principle, vulnerable to agents thatcause DNA damage. Thus a chemotherapeutic DNA damaging agent can be targeted to can‐cers that lack the capability to repair the particular type of DNA damage caused by theagent. This can lead to a level of DNA damages that overwhelms the defenses of the cancercells and causes their death. Non-cancerous cells with normal repair would not be targeted.Thus the toxicity of such DNA damaging agents to the treated patient would be limited. Adramatic example of such targeted therapy is the high cure rate of testicular cancer due to adefect in the ability of the cancer cells to repair DNA inter-strand cross-links, and the use ofcross-linking platinum compounds to kill such cells.

Another strategy, which is currently the basis for numerous ongoing clinical trials, involvessynthetic lethality. By this strategy cancers that are deficient in one DNA repair pathway canbe made more vulnerable to DNA damage by treatment with agents that inhibit an addi‐tional repair pathway. Promising clinical results, so far, have been obtained in the treatmentof patients with breast and ovarian cancer due to an inherited genetic defect in the homolo‐gous recombinational repair pathway. Such cancers are deficient in the ability to repair dou‐ble-strand breaks. Treatment of these cancers with an agent that interferes with anotherpathway that ordinarily repairs single-strand breaks allows such breaks to accumulate andto be converted to double-strand breaks during DNA replication. The increase in double-strand breaks appears to overwhelm the cancer cells, while sparing normal cells, thus pro‐viding positive clinical benefit to the patient without much toxicity.


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Author details

Carol Bernstein1*, Anil R. Prasad2, Valentine Nfonsam3 and Harris Bernstein4

*Address all correspondence to: [email protected]

1 Research Service Line, Southern Arizona Veterans Affairs Health Care System, Tucson,AZ, USA

2 Department of Pathology, University of Arizona, Tucson, AZ, USA

3 Department of Surgery, University of Arizona, Tucson, AZ, USA

4 Department of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA

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